title: Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.
abstract: Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research.
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Camden, NSW, Australia.
authors.forename: Ratna B
authors.initials: RB
authors.lastname: Gurung
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Camden, NSW, Australia.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Begg
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Camden, NSW, Australia.
authors.forename: Auriol C
authors.initials: AC
authors.lastname: Purdie
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Camden, NSW, Australia. Electronic address: richard.whittington@sydney.edu.au.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Whittington
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics; D019540:Area Under Curve; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D018985:Epitopes, B-Lymphocyte / Q000235:genetics; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000276:immunology*; D010283:Paratuberculosis / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D010865:Pilot Projects; D015203:Reproducibility of Results; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000276:immunology / Q000382:microbiology*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D018985:Epitopes, B-Lymphocyte
keywords: ELISA; Paratuberculosis; Proteins; Stress; Synthetic peptides